Post-translational modification by the Pgf glycosylation machinery modulatesStreptococcus mutansphysiology and virulence

AbstractStreptococcus mutansis a keystone pathogen of dental caries, and the ability to form biofilms is essential for its pathogenicity. We identified a glycosylation machinery (Pgf) inS. mutansthat post-translationally modifies two surface-associated adhesins, Cnm and WapA. The fourpgfgenes (pgfS,pgfM1,pgfE,andpgfM2) are part ofS. mutanscore genome and we hypothesized that the scope of Pgf goes beyond Cnm and WapA. By inactivating eachpgfgene individually or creating a quadruplepgfmutant inS. mutansOMZ175, we showed that the Pgf machinery is important for biofilm formation. Compared to OMZ175, differences in surface charge, membrane stability, and genetic competence were also observed for most mutants. Importantly,in silicoanalyses and tunicamycin MIC assays suggest a functional redundancy between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Using a rat oral colonization model, we showed a 10-fold reduction in recovered CFUs for thepgfquadruple mutant compared to OMZ175. Finally, using Cnm as a model, we showed by glycoproteomics analyses that Cnm is heavily modified with N-acetyl hexosamine in OMZ175 whereas phosphorylations were observed for thepgfSmutant. Our findings indicate that the Pgf machinery participates in important aspects ofS. mutanspathobiology.Graphical AbstractAbbreviated summaryIn this study, we demonstrate that the Pgf glycosylation machinery ofStreptococcus mutans, a keystone pathogen of dental caries, regulates several aspects of bacterial pathophysiology that ultimately contribute toS. mutansfitness in oral colonization experiments. Using the heavily glycosylated Cnm adhesin as a model, we found that inactivation of the glycosyltransferase PgfS results in loss of Cnm glycosylation, but instead, Cnm became heavily phosphorylated, suggesting a crosstalk/competition between these two post-translational modification mechanisms.